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Resumen Introducción: El síndrome de ojo seco es una enfermedad en la que se generan signos y síntomas que conducen a alteraciones oculares prolongadas, por lo tanto, es relevante establecer con precisión la etiología de la enfermedad con la finalidad de establecer el tratamiento más efectivo, de allí, la importancia del desarrollo de exámenes innovadores como son los biomarcadores, los cuales permiten identificar con mayor precisión el cuadro clínico. Por esta razón, el presente trabajo pretende describir los principales avances de los biomarcadores de la superficie ocular y reconocer su aplicación clínica para el diagnóstico de ojo seco entre los años 2013 a 2018. Metodología: Se analizó literatura sobre biomarcadores empleados para el diagnóstico del ojo seco, mediante una revisión sistemática tipo narrativa de 2013 a 2018 por medio de los descriptores controlados "Dry Eye Syndrome" "biomarkers" "tear proteins" "eye proteins" seleccionados en DeCS y Pubmed; la búsqueda arrojó 48 estudios, de los cuales seleccionamos 21 para el análisis. Resultados: Son diversas las proteínas lagrimales que pueden ser relacionadas con la presencia y ausencia de la enfermedad, es vital que los biomarcadores sean valorados como una herramienta alternativa para diagnosticar con facilidad y precisión la enfermedad del ojo seco. Discusión: Los biomarcadores permiten reconocer los procesos patógenos y biológicos del síndrome de ojo seco, al reflejar el estado de la superficie ocular en presencia o ausencia de signos y síntomas, facilitando el diagnóstico precoz, seguimiento, tratamiento y control de la enfermedad.
Abstract Introduction: Dry eye syndrome is a disease in which signs and symptoms that lead to prolonged ocular alterations occur, therefore, it is relevant to accurately establish the etiology of the disease with the configuration of establishing the most effective treatment, hence the development of innovative exams such as biomarkers selected with greater precision the clinical picture. For this reason, the present work aims to describe the main advances of biomarkers of the ocular surface and to recognize their clinical application for the diagnosis of dry eye between 2013 and 2018. Metodology: Literature on biomarkers used for the diagnosis of dry eye was analyzed, by means of a systematic narrative review from 2013 to 2018 by means of the controlled descriptors "Dry Eye Syndrome" "biomarkers" "tear proteins" "eye proteins" selected in DeCS and Pubmed; The search yielded 48 studies and 21 studies were selected for the analysis. Results: There are several tear proteins that can be related to the presence and absence of the disease, it is vital that biomarkers are evaluated as an alternative tool to easily and accurately diagnose dry eye disease. Discussion: Biomarkers allow to recognize the pathogenic and biological processes of dry eye syndrome, reflecting the state of the ocular surface in the presence or absence of signs and symptoms, facilitating early diagnosis, monitoring, treatment and control of the disease.
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Humans , Biomarkers , Dry Eye Syndromes , Cytokines , Matrix Metalloproteinases , Lacrimal Apparatus , MucinsABSTRACT
Background Retinitis pigmentosa (RP) is one of the causes of congenital blindness.It is well known that the degeneration process of rod cells is difficult to detect in RP.Retinal degeneration 12 (rd12) mice is a new,spontaneously arising mouse model for human Leber congenital amaurosis (LCA),and it is helpful for us to explore the pathogenesis and determine the treating target of RP.Objective This study was to investigate the natural disease process of short-length sensitive cone cells in rd12 mice,a LCA Rpe65rd12 (B6 [A]-Rpe65rd12/J) mouse.Methods The rd12 mice at postnatal (P) 14,P21,P35 and P90 were selected (5 mice for each),and the wild-type C57BL/6J (B6) mice with matched ages were included as controls.Photopic full-field electroretinogram (ERG) was recorded with Roland Q450SC UV visual physiology instrument.Cone response was recorded using single white light-emitting diode (LED) stimulation with the flash intensity of 1.00 cds/m2 and 1.96 cds/m2,and short wave-length sensitive cone response was recorded using ultraviolet light ([363 ±6] nm) stimulation with the flash intensity of 2.0 mWs/m2 and 3.0 mW/m2.The mice were sacrificed and retinal whole-mounts were prepared.The distribution and number of cone cells and UV-sensitive cone cells were detected by FITC-peanut agglutinin (FITC-PNA) and Cy3 immunofluorescence stainning,respectively.Results In P14 rd12 mice,the ERG responses of overall cone cells presented the negative waveform and the latency was delayed,and UV-sensitive cone response was unrecordable.The b-wave amplitude of overall cone cells reduced by 75％ in P21 rd12 mice compared with wild-type B6 mice,and the mean latency of b-wave in the P21 rd12 mice was significantly longer than that in the wild-type B6 mice ([102.80± 11.39] ms vs.[43.40± 5.60] ms) (t =-8.106,P =0.001).The mean b-wave amplitudes of U Vsensitive cone cells were (59.60± 36.00),(82.40± 12.22) and (68.43 ± 17.63) μV in the wild-type B6 mice,andthose in the rd12 mice were unrecordable.Immunofluorescence showed that a lager number of cone cells with green fluorescence were seen,and the expression of opsin with red fluorescence was displayed in the UV-sensitive cone cells of nasal lateral on retinal ventral side in P14 wild-type B6 mice;while only a few opsin positive-response cells were seen in P14,P21 and P35 rd12 mice.Conclusions In rd12 mice,the functional abnormality and quantitative reduction of cone cells appear in the early postnatal days,and the loss of UV-sensitive cone cells is earlier and more obvious.
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Background Statins has prominent roles in regulating lipids,anti-inflammation,autoxidation and protecting vascular endothelial cells.Sartans can promote cell growth and the expression of cytokines.Since the pleiotropic effects of statins and sartans on a variety of cell types,it is inferred that the two medicines can delay retinal aging.Objective This study was to explore the anti-aging effect of simvastatin and telmisartan on the physiological aging of retina.Methods Sixty-six three-month-old healthy SD rats were selected in this study,and 6 of them served as the youth group and the right eyeballs were immediately enucleated.The other rats were raised until 9-month-old in the same conditions and then randomly divided into the simvastatin group,telmisartan group and the control group with 20 rats for each group.The simvastatin of 5 mg/kg and telmisartan of 8 mg/kg were given by intragastric administration once a day in the simvastatin group and the telmisartan group until 17-month-old,and the equal amount of normal saline was used in the control group in the same way.The number of survival rats was 12 in the simvastatin group,10 in the telmisartan group and 8 in the control group.The right eyes were enucleated after heart perfusion of 4％ paraformaldehyde solution for the preparation of retinal paraffin sections.Retinal thickness was measured by pathological examination,and the expressions of the retinal neuron markers,including Thy-1,protein kinase C-α (PKC-ot),opsin and rhodopsin,were detected by immunofluorescence technique to evaluate the morphology of retinal ganglion cells (RGCs),bipolar cells as well as the thickness of the outer segment of photoreceptors.Results The retinal structure was clear in the rats of the youth group.However,the RGCs arrangement and inner segment (IS) and outer segment (OS) structure were abnormal in the simvastatin group,the telmisartan group and the control group.Compared with the rats of the youth group,the thickness of outer nuclear layer (ONL),outer plexiform layer (OPL),inner nuclear layer (INL),inner plexiform layer (IPL) and the total thickness of the aging rats were decreased,and the IS/OS thickness was increased in the simvastatin group and the telmisartan group (all at P＜ 0.01).Thy-1 stain showed that the number of RGCs was reduced in the simvastatin group,telmisartan group and the control group compared with the youth group,and that in the simvastatin group was increased in comparison with the control group (all at P＜0.01).PKC-αt stain exhibited that the density of bipolar cells was increased but the axon terminal bouton was declined in the simvastatin group,telmisartan group and the control group compared with the youth group,and the axon terminal bouton was declined in the simvastatin group compared with the youth group and the control group (all at P=0.000).Opsin and rhodopsin stains displayed that the OS thickness was increased in the simvastatin group,telmisartan group and the control group compared with the youth group,and that in the telmisartan group was reduced in comparison with the control group (all at P＜0.01).Conclusions As SD rat aging,retinal thickness is gradually attenuated and the number of RGCs is gradually declined.Although the density of bipolar cells seem to be unchanged,their synaptic connections are decreased and the OS is thicken.Simvastatin and telmisartan can delay retinal senescence by protecting retinal neurons against aging and thinning thickened OS.
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Background Age-related cataract is a common cause of blindness.However,its cause and pathogenic mechanism have not been fully understood.Recent studies revealed that aquaporin 1 (AQP1) and AQP0 are closely related to the pathogenesis of cataract.Objective This study was to investigate the differential distribution and expression of AQP0 and AQP1 in lenses with age-related cataract and explore its effect on pathogenesis of age-related cataract.Methods Seventeen anterior capsular membrane samples and nucleus samples of lenses were collected from age-related cataract patients during the small incision nonphacoemulsification cataract extraction,and 6 normal lens samples were obtained from health donors in the First Affiliated Hospital of Fujian Medical University.The expression and distribution of AQP1 and AQP0 in the lenses were detected by immunohistochemistry,and the relative expression levels of AQP1 and AQP0 proteins in the lenses were assayed by using Western blot assay.This study protocol was approved by Ethic Committee of this hospital,and written informed consent was obtained from each patient.Results Immunohistochemistry showed that in the normal lenses,AQP1 expressed mainly in LECs;while AQP0 primarily expressed in fiber cells of the lens cortex and nucleus.The relative expression levels of AQP1 and AQP0 in the lenses with age-related cataract (absorbance) were 0.223±0.008 and 0.118±0.015,which were significantly lower than 0.246±0.007 and 0.149±0.007 in the normal lenses (t =-4.508,-3.291,both at P＜0.01).Western blot revealed that the relative expression levels of AQP1 and AQP0 in the lenses with age-related cataract (absorbance) were 0.663 ± 0.012 and 0.599 ± 0.015,which were significantly reduced in comparison with 0.844±0.041 and 0.955 ±0.064 in the normal lenses (t =-7.492,P＜0.05;t =-9.570,P＜0.01).Conclusions AQP1 and AQP0 distribute in different sites of lenses.The expressions of AQP1 and AQP0 are obviously down-regulated in lenses with age-related cataract,suggesting that AQP1 and AQP0 probably play different roles in the pathogenesis of age-related cataract.
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Tear fluid is a complex body fluid,which may contain thousands of protein/peptides and other molecules.Studies have determined that the changes in the chemical compositions of tears play an important role in some diseases and their progression.Tear fluid is a useful and easily accessible source for understanding ocular surface diseases,other eye diseases,and systemic diseases.It can also be used for identification of biomarkers for clinical applications and pharmaceutical development.Therefore,quantitative proteomic analysis of tears may provide very important information for diagnosis and treatment of eye diseases or the development of new drugs.Knowledge of the current proteomic technologies for tear analysis is helpful for conducting studies.In addition,ophthalmologists should pay close attentions to the association between tear proteomic changes and eye diseases,recent advances in tear proteomics and their applications in studying ocular surface diseases and conditions.
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Objective To investigate the effect of pigment epithelium-derived factor (PEDF) on the pathogenesis of preeclampsia disease,by detecting the expression of PEDF in the placentas,as well as the relationship between PEDF and the production of placental vessels.Methods A study was performed in 60 cases of pregnant women with preeclampsia in the obstetrical department of Nanfang Hospital affiliated to southern medical university from October 2011 to January 2013,in which 30 cases were patients with mild preeclampsia(mPE) and other 30 cases were those with severe preeclampsia (sPE).40 normal pregnant women who also been hospitalized and delivered were selected as control group.The expression of PEDF and micro-vessel density (MVD) in placentas were assayed by using western blot and SP immunohistochemical method,then the relationship between PEDF and MVD was analyzed.Results (1) The pathological changes of placentas:the placental weight were lightened obviously in the mild preeclampsia and severe preeclampsia groups,the reduced blood vessels and luminal stegnosis were found in chorionic villus,basement membrane of trophocytes were thickening.The hyperplasia syneytiotrophoblast were like nodosity,with focus infarction,fibrinoid necrosis,or thrombogenesis.While there was no the above mentioned pathological alteration in normal control group.(2)The levels of PEDF expression in mild and severe preeclampsia group were 0.63 ± 0.09,0.93 ± 0.07,while 0.47 ± 0.04 in control group,which in mild and sever preeclampsia groups were significantly higher than that in normal group (P ＜ 0.05).Compared to mild preeclampsia group,the expression of PEDF was significantly increased in severe preeclampsia group,there was statistical significance between the difference (P ＜ 0.05).(3) The amount of microvessel density (MVD) in mild and severe preeclampsia group were 106 ±9,93 ±8,while 136 ±9 in control group,which were significantly reduced in mild and severe preeclampsia group,compared to that in normal control group (P ＜ 0.05).And it was significantly lower in severe preeclampsia group than that in mild preeclampsia group (P ＜ 0.05).(4) The expression of PEDF was negatively correlated with the amount of MVD in mild and severe preeclampsia group (r =-0.426,P ＜ 0.05 ; and r =-0.646,P ＜ 0.05 respectively),which was also negative in control group (r =-0.589,P ＜ 0.05).Conclusion Increased PEDF expression in placentas of women with preeclampsia induce the dysfunction of the placental vascular reconstruction and the pathological alteration like ischemic and hypoxia in placentas,which may be involved in pathogenesis and pathogenic progress of preeclampsia.
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ObjectiveTo analyze the status of DACH1 gene promoter methylation and explore its association with the expression of DACH1 gene promoter methylation and clinical significance of endometrium carcinoma(EC).Methods From February 2004 to August 2008,a total of 80 EC tissue samples with comprehensive surgical pathology staging were collected and used for this study.Twenty normal endometrium tissues in 2008 were abtained from the fractional curettage because of dysfunctional uterine bleeding as control.All samples were confirmed pathologically.Methylation specific PCR (MSP) was performed to detect the promoter methylation of DACH1 gene,and analyze its influence on the expression of DACH1 and the relationship between DACH1 promoter methylation and clinicopathological factors in EC.DACH1 protein expression was detected by western blot.Chi-square test and Pearson test were used for statistical analysis.ResultsThe rate of promoter methylation of DACH1 gene in the EC tissues was significantly higher than that in the normal endometrium issues (30％ vs.5％,P ＜ 0.05).There was an association between the expression of DACH1 and DACH1 gene promoter methylation ( r =- 0.30,P ＜ 0.01 ).There was statistical difference between the methylation of DACH1 and the pathological grade ( P ＜ 0.05 ) or histological type ( P ＜0.05).But DACH1 gene methylation was not related with the age,stage,myometrial invasion depth and lymphnode metastasis (P ＞ 0.05 ).Conclusions DACH1gene promoter methylaion could lead to a decrease or absence in the DACH1 expression in EC.The promoter methylation of DACH1 gene may induce the inhibition of DACH1 expression,which might be one of the mechanisms of DACH1 gene inactivation in human EC.
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Objective To construct eukaryotic expression plasmid pIRESneo3-pigment epithelium-derived factor (PEDF) and detect its transient expression in SP2/0 cells. Methods Specific primers were designed based on the mature peptide sequence of human PEDF cDNA in the GenBank. Human PEDF gene was cloned into the eukaryotic expression vector pIRESneo3. The PEDF DNA was transfected into SP2/0 with LipofectamineTM 2000. The recombinant human PEDF protein expressed in SP2/0 cell culture supernatant was identified by Western blot and enzyme-linked immunosorbent assay. The biological activity of the recombinant human PEDF was measured by 3-(4,5-dimethylthiazol-z-y1)-2,5-diphenytetrazolium bromide(MTT) method. Results PCR amplification, restriction enzyme digestion and DNA sequencing confirmed that the mature peptide sequence of human PEDF cDNA was successfully cloned into the eukaryotic expression vector pIRESneo3. And the plasmid was transfected into SP2/0 cells, which could secret PEDF. Western blot analysis showed that there was only one obvious band at the position of relative molecular weight of 50 000, and it is equivalent to the expected value. Enzyme-linked immunosorbent assay suggested that the content of PEDF began to rise after transfection, and peaked at 36 h [(0.92±0.04) μg/ml]. The proliferation of human umbilical vein endothelial cell line was significantly inhibited by supernatant after transfection of 36 h (P<0.05). Conclusions The eukaryotic expression plasmid pIRESneo3-PEDF had been successfully constructed and active human PEDF was transiently secreted, which made a foundation for further study of stable expression and purification of PEDF. This protein could be a potential medication for preventing and managing retinopathy of prematurity.
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Objective To screen the mutations of MYOC gene in a Chinese primary open angle glaucoma (POAG) family from Cbengqing and investigate the relationship between the mutations in MYOC/TIGR gene and POAG.Methods In a large 4-generation glaucoma family, myocilin gene (MYOC) was screened in 39 family members, 8 of which were confirmed patients. Normal controls included 100 normal Chinese subjects.The known mutations of MYOC gene ( including G34C, C136T, G144T, G227A, C624G,G736A, C1009G, A1036G, C1081T, G1099A, G1138A, A1139C, T1430A, C1441A and C1442T) were detected by single strand conformation polymorphism(SSCP) , po]ymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis and DNA sequencing.Results G227A mutation was detected in 2 POAG patients and 1 asymptomatic patient, but not in the controls.Cl009del mutation was identified in all patients of the pedigree and an offspring member but not in the controls. No other mutations were detected.Since the C1009del mutation was revealed for the first time, a new GenBank number FJ237047 correponding to ACI62293 was applied.Conclusions The G227A mutation is a known site and there is no relationship between G227A mutation and glaucoma. But C1009del may be related to glaucoma which suggests that morbidity could be higher in the relatives of POAG than the controls.
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Objective To assess the impact of pigment epithelium-derived factor(PEDF) on the migration of the glioma cells.Methods PEDF(100 ng/ml)was added to U87 cells(U87PFDF),and VEGF of 0.25μg/ml was added to U87PEDF+VEGF while U87 cells as control(U87con).The cell migratory assav was used tbr evaluating its inhibitory migration rate of PEDF.Real time RT-PCR was used for evaluating the expression of Laminin-8 gene.Results The number of migratory cells was higher than those with added PEDF,and the inhibitory rate of migratory was 38% even in the presence of VEGF.Real time RT-PCR revealed that the mRNA expression levels of α4,β1,γl were(1.043±0.090),(0.823±0.012).(0.762±0.05) copy/μl,which were higher than those treated by PEDF(0.633±0.004),(0.442±0.005).(0.424±0.002)copy/μl,respectively (P＜0.05).Conclusion PEDF could decrease the migmtory capacity of the glioma cells even in the presence of VEGF because of the regulation of Laminin-8 in part.
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Objective To assess the impact of pigment epithelium-derived factor(PEDF)on the proliferation and apoptosis of the glioma cells by detecting expression of apoptosis related proteins.Methods U87 cells were treated with PEDF(1000μg/ml,U87PEDF),or without PEDF(U87com0),cell proliferation assays were performed by MTT assay to test the effect of PEDF on proliferation of glioma cells;Apoptosis assays were performed by flow-cytometric analysis;Western-blot Was used for evaluating the expression of p16 protein.Results The induced inhibitony rates of glioma cells by PEDF were(54.29±0.62)% Compaxed with the control(t=2.63,P<0.05).The apoptosis assay showed that(21.84±0.36)% of PI- negative/annexin V-positive Was present in the U87 PEDF cells.The appoptosis was associated with the incteases of p16 protein(0.82±0.09)compared with tlle control(0.43±0.03,P<0.05).Conciusion PEDF may play a significant role in apoptosis regulation and proliferation of glioma cell accompanied with the increase of the p16 protein.